The use of hybrid plants in agricultural crops is well known as a means to enhance plant production and value. A hybrid plant is one resulting from a crossing (an outcrossing) other than a self-crossing (selfing) or a sibling-crossing (sibbing), in particular between inbred lines. Hybrid crossing, or hybridization, is commonly accomplished by having side by side stands or rows ("crossing" rows), with female stands (P1) alternating with pollinating rows (P2). Pollen is normally carried by insects or wind, although in some commercial plants pollination is by hand.
The value of the hybrid is primarily in the increased yield and vigor, or heterosis, which is displayed as compared to the parents, in particular in the first generation (F1) of progeny. Selfing of a hybrid (F1) results in a second generation of plants (F2) which normally exhibits less heterosis than is displayed by the F1 generation. Hybrids have been commercially used in a great variety of plants and crops, including wheat, field corn, sweet corn, barley, sorghum, sugar beets, garden beets, onions, tomatoes, cabbage, cauliflower, broccoli, brussels sprouts, cucumber, carrot, spinach, summer and winter squash, asparagus, pepper, eggplant, radish, muskmelon, watermelon, pumpkin, cantaloupe, tobacco and various ornamentals.
In developing hybrid seed (i.e., seed from which the first generation hybrid is to be grown), the ability to control mass pollination is important. Specifically, prevention of parental selfing or sibbing, which would result in the production of non-hybrid seed, is important. Several methods have been used, or considered, for ensuring or maximizing hybridization.
One method is to emasculate the female parent line (P1), either by hand or mechanically. For instance, in the alternating stand situation, if the P1 stand is emasculated, plants in that stand can only be pollinated by plants from another stand, i.e., the pollinating stand. While this has been done with some plants, e.g., tomato (manual) and corn (manual and mechanical), manual emasculation is labor intensive and impractical for volume crops, and mechanical emasculation cannot be used for many crops and can cause yields to suffer due to plant damage.
A second method is the use of chemical agents (gametocides) to emasculate the intended female parents. Although attempts have been made to develop such chemicals, the approach has a number of problems. The chemical must provide sufficient male sterility without otherwise affecting the plant. There is the need to treat selectively only the ultimate female parents and to spray only in the proper amounts. Plants sprayed with insufficient chemical or sprayed at the wrong stage of development may not be rendered male sterile (so-called "shedders"). In addition, the cost of the gametocide may be a factor.
A third method applies to those plants in which cytoplasmic male sterility (cms) has been found, e.g., cotton, tobacco, rice, corn, onions, sorghum, carrot, radish, alfalfa and certain flowers and grasses. For such plants, cms in the female parent will ensure its outcrossing. The approach is limited in that, in general, the occurrence of cms in crops is rare. Also, for those crops in which the product depends on fertilization the use of cms is workable only if a restorer gene is available for the particular plant to permit restoration of fertility of the F1 hybrid. In addition, cms is often associated with undesirable traits, some of which may not appear at the outset. For instance, a cms system used in maize led to the Southern leaf blight epidemic of the early 1970's. U.S. Pat. No. 2,753,663 discloses the use of cms in corn.
A fourth method which has been considered is the use of nuclear male sterility, also referred to as chromosomal or genic male sterility. Nuclear male sterile genes presumably result from a mutation at some point in the development of a plant or its ancestors. The genes occur in practically all diploid plant species which have been examined for this property, and are present as well in polyploid plants. The genes are normally recessive. Nuclear male sterile genes have been studied in a large number of plants of agricultural interest, e.g., wheat, corn, tomato, barley, pepper, rice, lima beans, peas, cotton, watermelon, soybeans, tobacco, lettuce and various ornamentals.
See R. Frankel et al., Pollination Mechanisms, Reproduction and Plant Breeding, (Springer Verlag), 3.4.1.2 and 3.4.2.1 (1977) See W. Gottschalk et al., "Induced Mutations in Plant Breeding", Monographs on Theoretical and Applied Genetics, 7, 70 (Table 13) (1983) for a list of induced male sterile mutants in cereals and dicotyledonous plants. For many plants there are a number of male sterile loci, each of which can have either a recessive male sterile (ms) or a dominant male fertile (Ms) allele. For a diploid plant, such a locus (allele pair) can be homozygous male sterile (ms/ms), homozygous male fertile (Ms/Ms), or heterozygous male fertile (Ms/ms). The nomenclature shown is for recessive male sterility. In the less likely instance of dominant male sterility, the nomenclature would be the opposite (Ms male sterile; ms male fertile). Unless otherwise stated, the nomenclature used herein is for recessive male sterility. Nuclear male sterility and nuclear male sterile plant, line, gene, locus, etc. shall on occasion be referred to herein, respectively, as male sterility and male sterile plant, line, gene, locus, etc.
Nuclear male sterility has use in hybrid breeding in that if the female parent displays nuclear male sterility (e.g., in a diploid plant, if the plant is homozygous for recessive ms), the female parent cannot function as a male parent and seeds obtained from it by outcrossing are necessarily hybrid seeds. In the alternating stand situation, nuclear male sterile female stands alternate with pollinating stands. Seed harvested from the female stands can be used as hybrid seed.
The use of nuclear male sterility in hybrid breeding requires a means to maintain the nuclear male sterile line. Since male steriles produce no pollen, to be maintained they must be produced anew each generation from segregating progenies. For a diploid plant, a maintainer line is typically obtained by crossing the male sterile line (ms/ms) with a male fertile line (Ms/Ms). The product is a heterozygous maintainer line (Ms/ms) which is used to generate the next generation male sterile line in a crossing to a homozygous male sterile line (ms/ms). The progeny of this crossing are 50% homozygous male sterile (ms/ms) and 50% heterozygotes (Ms/ms). In such a situation there must be a means to isolate the male sterile plants for use as the female parent in hybrid breeding. This has to a large extent been done by visual inspection, although there have been suggestions for using either natural linkage or chromosomal abnormalities to assist in isolating male steriles. See, in general, J. R. Welsh, Fundamentals of Plant Genetics and Breeding, Chaps. 16, 18 John Wiley and Sons, (1981) for a discussion of hybrid breeding and male sterility systems.
The visual inspection approach to isolating male steriles involves examining individual plants in the field at the time of anthesis. Male fertiles often show physical differences, e.g., in anther structure, which permit them to be mechanically or manually removed (rogued). While this approach has been used for some crops, it is not effective for many plants of agronomic interest. Moreover, it is labor intensive and cannot be used for selection at an early stage of plant development.
Another approach to obtaining male sterile stands involves the use of naturally occurring linkage. As used herein, the term linkage means genetic linkage between two or more loci (or genes) in a given genome such that the recombination frequency between the loci (or genes) is less than 50%, the frequency of random assortment. As so used, the term usually refers to loci (or genes) in sufficiently close proximity on the same chromosome of a given plant that recombination between them occurs less frequently than if the genes were located on separate chromosomes. The term, as used herein, also embraces other forms of genetic linkage where the recombination frequency deviates from random assortment. The extent of linkage is measured in terms of the recombination frequency, or recombination units. Linked genes are normally transmitted or inherited together at a frequency related to their recombination frequency, as can be shown by segregation analysis of progeny resulting from a crossing. If a chromosomal recombination (a crossover or other rearrangement event) takes place, linked alleles may not be transmitted together. The tighter the linkage, the less likely is a recombination resulting in disruption of the linkage. For instance, very tight linkage exists if the linked genes are adjacent to each other on the same chromosome. As stated, the extent of linkage is measured in terms of recombination units. If two genes are separated by ten recombination units, there is a 10% chance of their becoming uncoupled in a crossing. A separation of five recombination units constitutes a tighter linkage.
There have been a number of proposals for using naturally occurring linkages between a male sterile gene and a marker gene (i.e., linkages, involving male sterile mutants, either discovered in nature or resulting from a breeding program) as a means of detecting or isolating male sterile plants or seeds from a mixture of male steriles and male fertiles. There is a 1930 disclosure of the possibility of using naturally occurring linkage between nuclear male sterility and seed color in bicolor corn in a program to produce hybrid corn seed; W. R. Singleton et al., Journal of Heredity, 21, 266-68 (1930). Other disclosures concern the use of natural linkage in barley between male sterility and recessive DDT resistance as a means of removing male fertiles (upon application of DDT) in a hybrid program; G. A. Wiebe, Argon. J., 52, 181-82 (1960) and G. A. Weibe, Barley Newsletter, 8, 16 (1964). Wiebe (1964) also discloses the possibility of reducing the crossover value (that is, tightening the linkage) either by using radiation to invert chromosomal segments or by breeding for translocations. Use of linkage between a male sterility gene and a gene for non-germination upon treatment of a particular chemical is suggested in R. T. Ramage, Barley Newsletter, 9, 3-8 (1966). Linkage between male sterility and color, or lack-of-color, genes is disclosed in J. Philouze, Ann. Amel. Plant., 24, 77-82 (1974), cited in M. Yardanov, Monographs on Theoretical and Applied Genetics, 6, 189-219 (1983). Such linkage is also disclosed in C. A. Foster, Barley Genetics Newsletter, 9, 22-23 (1979). Linkage between a male sterile locus and a shrunken seed gene is disclosed in D. E. Falk et al., Barley Genetics, IV, 778-85 (1981). A tomato mutant with linkage between an isozyme marker and a male sterile locus resulting from a breeding program is disclosed in S. D. Tanksley, Plant Molecular Biology Reporter, 1, 3-8 (1983) and C. M. Rick et al., Isozymes: Current Topics in Biological and Medical Research, 1, 269-84 (1983). Natural linkage is also disclosed in C. A. Foster, Barley Genetics, III, 774-84 (1976); and R. T. Ramage, Monographs on Theoretical and Applied Genetics, 6, 71-93 (1983). The marker is termed a "haplo-viable" mutation (Ramage, at 84); it is linked to the dominant allele of the male sterile locus. So-called haplo-viable mutations can be transmitted through the egg but not through the pollen. The use of natural linkage to assist in isolating male steriles is limited by the lack of appropriate markers for many crops of agricultural interest and also, for those crops for which markers are known, by the lack of sufficient closeness of linkage.
Another approach for obtaining male sterile lines which has been suggested involves the use of chromosomal abnormalities. One type of abnormality is a differentially transmitted chromosome, e.g., a duplicate deficient chromosome (egg-viable but not transmitted by pollen) as disclosed in U.S. Pat. No. 3,710,511. This chromosomal variation is disclosed as occurring naturally or resulting from mutagenic agents. If a female stand is produced from a Ms/ms heterozygote in which the male fertile allele is linked with a differentially transmitted chromosome (transmitted through the female parent but not the male parent), selfing or sibbing can be avoided. Another type of chromosomal abnormality is the presence of an extra chromosome, meaning the presence of an additional chromosome from the same species or the presence of an additional chromosome from a related species. R. T. Ramage, Barley Newsletter, 9, 3-8 (1966) discloses a system in barley with an extra chromosome (a trisomic system) resulting from disjunction during mitosis. The extra chromosome is transmitted through the female germ line but only rarely transmitted through the male. Fertility genes and marker genes (e.g., phytocide susceptibility, height, seed size or shape) on the extra chromosome permit production and identification of male sterile female parents. C. J. Driscoll, Crop Sci., 12, 516-17 (1972) discloses an analogous system for hybrid wheat using an extra chromosome derived from rye. U.S. Pat. No. 4,051,629 also discloses an extra chromosome system. In addition, systems with extra chromosomes are disclosed in R. Frankel et al., Pollination Mechanisms, Reproduction, and Plant Breeding, (Springer Verlag), .sctn.3.4.4 (1977); P. Wilson et al., "Hybrid Wheat", Monographs on Theoretical and Applied Genetics, Chap. 4, 94-123 (1983); and J. Sybenga, Theor. Appl. Genet., 66, 179-201 (1983). Sybenga, at 194, suggests the further development of an extra ("alien") chromosome system by constructing such a chromosome using molecular genetic engineering. In general, the extra chromosome approach is limited (a) by difficulties in deriving the appropriate system, as well as (b) by problems of low pollen yield.